U.S. patent number 7,459,307 [Application Number 10/485,816] was granted by the patent office on 2008-12-02 for composition for treatment of articular cartilage damage.
This patent grant is currently assigned to Chul-Won Ha, Medipost Co., Ltd. Invention is credited to Chul-Won Ha, Sung-Eun Yang, Yoon-Sun Yang.
United States Patent |
7,459,307 |
Ha , et al. |
December 2, 2008 |
**Please see images for:
( Certificate of Correction ) ** |
Composition for treatment of articular cartilage damage
Abstract
Disclosed herein is a composition for the treatment of articular
cartilage damage or loss or defect. The composition for the
treatment of articular cartilage injury of the present invention
includes (i) cellular components separated, proliferated, and/or
differentiated from the umbilical cord blood, (ii) a culture
medium; and (iii) a biocompatible polymer. The composition has very
superior ability of proliferation and differentiation and easier to
be collected and acquired.
Inventors: |
Ha; Chul-Won (135-785 Seoul,
KR), Yang; Yoon-Sun (Seoul, KR), Yang;
Sung-Eun (Seoul, KR) |
Assignee: |
Medipost Co., Ltd (Seoul,
KR)
Ha; Chul-Won (Seoul, KR)
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Family
ID: |
36746258 |
Appl.
No.: |
10/485,816 |
Filed: |
August 14, 2002 |
PCT
Filed: |
August 14, 2002 |
PCT No.: |
PCT/KR02/01552 |
371(c)(1),(2),(4) Date: |
April 05, 2004 |
PCT
Pub. No.: |
WO03/015802 |
PCT
Pub. Date: |
February 27, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20040151703 A1 |
Aug 5, 2004 |
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Foreign Application Priority Data
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Aug 14, 2001 [KR] |
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2001-49147 |
Aug 14, 2001 [KR] |
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2001-49148 |
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Current U.S.
Class: |
435/325;
424/93.1; 424/422 |
Current CPC
Class: |
A61K
35/51 (20130101); A61P 19/04 (20180101); A61K
35/28 (20130101); A61P 19/02 (20180101); A61P
19/00 (20180101); Y02A 50/30 (20180101) |
Current International
Class: |
C12N
5/00 (20060101); A01N 63/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1099754 |
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May 2001 |
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EP |
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98/51317 |
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Nov 1998 |
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WO |
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Other References
Radice M et al. 2000. Hyaluronan-based biopolymers as delivery for
bone marrow-derived mesenchymal progenitors. J Biomed Mater Res 50:
101-109. cited by examiner .
Alejandro et al., "Mesenchymal progenitor cells in human umbilical
cord blood", British Journal of Haematology, Oxford, GB, vol. 109,
No. 1, Apr. 2000, pp. 235-242. cited by other .
Wu Chengru et al., China Journal Surgery, Feb. 2001, vol. 39, No.
2, pp. 144-147 (indicated in Office Action as vol. 29). cited by
other .
Caplan et al., "Principles of cartilage repair and regeneration",
Clinical Orthopaedics and Related Research, Sep. 1997, No. 342, pp.
254-269. cited by other .
M.F. Pittenger et al., Science, vol. 284, Apr. 2, 1999, pp.
143-147. cited by other .
H.M. Lazarus et al., Bone Marrow Transplantation, 1995, vol. 16,
pp. 557-564. cited by other .
F. Barry et al., Experimental Cell Research, 2001, vol. 268, pp.
189-200. cited by other .
N. Jaiswal et al., Journal of Cellular Biochemistry, 1997, vol. 64,
pp. 295-312. cited by other .
S. Nehrer et al., Biomaterials, 1998, vol. 19, pp. 2313-2328. cited
by other .
P.L. Fitzpatrick et al., Aust. N.Z. J. Surg., 1998, vol. 68, pp.
573-579. cited by other .
Cesare Campagnoli et al., "Identification of Mesenchymal
Stem/Progenitor Cells in Human First-Trimester Fetal Blood, Liver,
and Bone Marrow", Hematopoiesis, Blood, Oct. 15, 2001, vol. 98, No.
8, pp. 2396-2402. cited by other .
Sarah A. Wexler et al., "Adult Bone Marrow Is A Rich Source of
Human Mesenchymal `Stem` Cells But Umbilical Cord and Mobilized
Adult Blood Are Not", British Journal of Haematology, 2003
Blackwell Publishing Ltd., vol. 121, pp. 368-374. cited by other
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Sekiya, I., et al., "In vitro cartilage formation by human adult
stem cells from bone marrow stroma defines the sequence of cellular
and molecular events during chondrogenesis," PNAS, 99:7, 4397-4402,
Apr. 2, 2002. cited by other .
Sekiya, I., et al., "Expansion of Human Adult Stem Cells from Bone
Marrow Stroma: Conditions that Maximize the Yields for Early
Progentiors and Evaluate Their Quality," Stem Cells, 20, 530-541,
2002. cited by other .
Indrawattana, N., et al., "Growth factor combination for
chondrogenic induction from human mescenchymal stem cell," Biochem.
and Biophy. Research Comm., 320, 914-919, 2004. cited by other
.
Radice, M. et al., "Hyaluronan-based biopolymers as delivery
vehicles for bone-marrow-derived mesenchymal progenitors,"
Institute of Histology and Embryology, Univ. of Padova, Padova,
Italy, Clinic of Orthopedic and Trauma Surgery, Univ. of Padova,
Padova, Italy, accepted Aug. 4, 1999; pp. 101-109. cited by other
.
Grande, D.A., et al., "Repair of Articular Cartilage Defects Using
Mesenchymal Stem Cells," Tissue Engineering, vol. 1, No. 4, 1995,
pp. 345-353. cited by other .
Elisseeff, J., et al., "Transdermal photopolymerization for
minimally invasive implantation," Proc. Natl. Acad. Sci. USA, vol.
96, Mar. 1999, pp. 3104-3107. cited by other .
Saim, A.B., et al., "Engineering Autogenous Cartilage in the Shape
of a Helix Using an Injectable Hydrogel Scaffold," The
Laryngoscope, 110, Oct. 2000, pp. 1694-1697. cited by other .
Grande, D.A., et al., "Cartilage Tissue Engineering: Current
Limitations and Solutions," Clinical Orthopaedics and Related
Research, No. 367S, 1999, pp. S176-S185. cited by other .
Von der Mark, K., et al., "Relationship between cell shape and type
of collagen synthesised as chondrocytes lose their cartilage
phenotype in culture," Nature, vol. 267, Jun. 9, 1977, 531-532.
cited by other .
Benya, P.D., et al., "Dedifferentiated Chondrocytes Reexpress the
Differentiated Collagen Phenotype When Cultured in Agarose Gels,"
Cell, vol. 30, Aug. 1982, pp. 215-224. cited by other.
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Primary Examiner: Barnhart; Lora E
Attorney, Agent or Firm: Sughrue Mion, PLLC
Claims
What is claimed is:
1. A composition for the treatment of articular cartilage damage,
loss, or defect, said composition comprising mesenchymal stem cells
isolated from umbilical cord blood, a culture medium, and a
biocompatible polymer, wherein said composition treats articular
cartilage damage, loss, or defect when administered to a site of
articular cartilage damage, loss, or defect in a subject.
2. The composition for the treatment of articular cartilage damage
of claim 1, wherein the cellular component is contained in an
amount of 1.times.10.sup.6 to 5.times.10.sup.7 cells in 1 ml of the
composition.
3. The composition for the treatment of articular cartilage damage
of claim 1, wherein said biocompatible polymer biodegrades.
4. The composition for the treatment of articular cartilage damage
of claim 3, wherein said biocompatible polymer is at least one
selected from the group consisting of natural polymers and
synthetic polymers comprised of hydroxy acids.
5. The composition for the treatment of articular cartilage damage
of claim 4, wherein said biocompatible polymer is at least one
selected from the group consisting of fibrin, gelatin, collagen,
hyaluronic acid, polyphosphazine, polyacrylate, polyglactic acid,
polyglycolic acid, pluronic acid, alginic acid, and their
salts.
6. The composition for the treatment of articular cartilage damage
of claim 5, wherein said biocompatible polymer further comprises a
chitosan.
7. The composition for the treatment of articular cartilage damage
of claim 5, wherein said biocompatible polymer is pluronic acid in
a final concentration of 30% by weight based on the total volume of
the composition.
8. The composition for the treatment of articular cartilage damage
of claim 5, wherein said biocompatible polymer is the hyaluronic
acid in a final concentration of 3-4% by weight based on the total
volume of the composition.
9. The composition for the treatment of articular cartilage damage
of claim 5, wherein said biocompatible polymer is pluronic acid in
a final concentration of 15-30% by weight and hyaluronic acid in a
final concentration of 2-4% by weight, each based on the total
volume of the composition.
10. The composition for the treatment of articular cartilage damage
of claim 9, wherein said biocompatible polymer further comprises
chitosan.
11. The composition for the treatment of articular cartilage damage
of claim 3, wherein the said composition contains 1.times.10.sup.6
to 5.times.10.sup.7 cells per mL of the composition.
12. The composition for the treatment of articular cartilage damage
of claim 4, wherein said composition contains 1.times.10.sup.6 to
5.times.10.sup.7 cells per mL of the composition.
13. The composition for the treatment of articular cartilage damage
of claim 5, wherein said composition contains 1.times.10.sup.6 to
5.times.10.sup.7 cells per mL of the composition.
14. The composition for the treatment of articular cartilage damage
of claim 6, wherein said composition contains 1.times.10.sup.6 to
5.times.10.sup.7 cells per mL of the composition.
15. The composition for the treatment of articular cartilage damage
of claim 7, wherein said composition contains 1.times.10.sup.6 to
5.times.10.sup.7 cells per mL of the composition.
16. The composition for the treatment of articular cartilage damage
of claim 8, wherein said composition contains 1.times.10.sup.6 to
5.times.10.sup.7 cells per mL of the composition.
17. The composition for the treatment of articular cartilage damage
of claim 9, wherein said composition contains 1.times.10.sup.6 to
5.times.10.sup.7 cells per mL of the composition.
18. The composition for the treatment of articular cartilage damage
of claim 10, wherein said composition contains 1.times.10.sup.6 to
5.times.10.sup.7 cells per mL of the composition.
19. The composition for the treatment of articular cartilage damage
of claim 4 wherein said natural polymer is a protein or
polysaccharide.
20. A method for treatment of articular cartilage damage, loss, or
defect in a subject in need thereof comprising administering the
composition of claim 1 to a site of articular cartilage damage,
loss, or defect in said subject.
21. A method for treatment of articular cartilage damage, loss, or
defect in a subject in need thereof comprising administering the
composition of claim 2 to a site of articular cartilage damage,
loss, or defect in said subject.
22. A method for treatment of articular cartilage damage, loss, or
defect in a subject in need thereof comprising administering the
composition of claim 3 to a site of articular cartilage damage,
loss, or defect in said subject.
23. A method for treatment of articular cartilage damage, loss, or
defect in a subject in need thereof comprising administering the
composition of claim 4 to a site of articular cartilage damage,
loss, or defect in said subject.
24. A method for treatment of articular cartilage damage, loss, or
defect in a subject in need thereof comprising administering the
composition of claim 5 to a site of articular cartilage damage,
loss, or defect in said subject.
25. A composition for the treatment of articular cartilage damage,
loss, or defect, said composition comprising mesenchymal stem cells
isolated from umbilical cord blood, a culture medium, and a
biocompatible polymer, wherein the composition contains
1.times.10.sup.6 to 5.times.10.sup.7 cells per mL of the
composition; wherein the biocompatible polymer is hyaluronic acid
in a final concentration of 0.5% -4% by weight based on the total
volume of the composition; and wherein said composition treats
articular cartilage damage, loss, or defect when administered to a
site of articular cartilage damage, loss, or defect in a
subject.
26. The composition of claim 25, wherein the final concentration of
hyaluronic acid is 1-4% by weight based on the total volume of the
composition.
27. The composition of claim 25, wherein the final concentration of
hyaluronic acid is 1.5-4% by weight based on the total volume of
the composition.
28. The composition of claim 25, wherein the final concentration of
hyaluronic acid is 2-4% by weight based on the total volume of the
composition.
29. The composition of claim 1, which is an injectable
formulation.
30. The composition of claim 3, which is an injectable
formulation.
31. The composition of claim 4, which is an injectable
formulation.
32. The composition of claim 5, which is an injectable
formulation.
33. The composition of claim 25, which is an injectable
formulation.
Description
This is a National Stage application under 35 U.S.C. .sctn. 371 of
PCT/KR02/01552 which claims benefit of Korean Patent Applications
2001/49147 and 2001/49148 filed on Aug. 14, 2001, all of which are
incorporate herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to a composition for the treatment
of articular cartilage damage or loss or defect.
2. Description of the Prior Art
The articular cartilage damage induces pain in the articular
region, fault of the articular movement, etc., and lowers the
quality of life as well as productivity. Particularly, it is
difficult to treat completely the articular cartilage damage since
the natural healing power is very low and it is connected to the
damage to the entire articulation as it is progressed continuously
once it occurs.
The methods of treatment of articular cartilage damage developed up
to the present time include chondroplasty, osteochondral,
transplantation, autologous chondrocyte transplantation, etc.
Chondroplasty is the most generally used method among the
above-described methods. The arthroscopic operation which is the
representative method is a method in which diagnosis and operation
may be performed simultaneously while magnifying and observing
inside of the articulation through a TV monitor by inserting an
arthroscope, on which a small-sized camera is mounted, into the
articular cavity through a small hole of less than 1 cm.
The above method is advantageous in that it is possible to reduce
pain and burden of a patient since it is not necessary to have a
direct incision of articulation and it is possible to cure
immediately after minor damages to tissues are observed through the
arthroscope. However, this chondroplasty is not satisfactorily
effective in view of its functional aspects since the
fibro-cartilage, not hyaline cartilage that is necessary for the
articulation actually, is produced mainly.
In the meantime, osteochondral transplantation is a method of
producing hyaline cartilage by collecting both of the cartilage and
sub-cartilage portions produced already in the normal section of a
patient and transplanting them in the damaged cartilage section by
making holes properly. This method has been successful in some
patients. However, this method may not be said to be a perfect
treatment method because of a problem of having cracks between the
transplanted portion and original tissues, and is not a general
method in that it may be applied to only the patients who are able
to be subject to autologous transplantation. And the above method
may not be operated if the damaged portion is large since the
donated portion is limited, and it is likely that complications
occur in the donated portion. Also, the process of operation is
comparatively complicated, and sometimes it is not possible to
perform arthroscopic operation. In other words, the above method
has weaknesses such as a new pain in the donated portion and may
incur complications such as a slow rehabilitation including pain,
fracture, bleeding, and scar after operation.
Autologous chondrocyte transplantation that has been started to be
employed recently is a method of filling the cartilage portion
damaged by proliferation of these cells by obtaining chondrocytes
from cartilage tissues collected from the normal portion of a
patient, culturing and growing them as much as they are needed
externally, securing a space by using periosteum and injecting them
to the damaged portion of cartilage along with the culture
medium.
Compared to the osteochondral transplantation method in which
already produced cartilage tissues are injected to the damaged
portion, there is a more possibility of reproducing hyaline
cartilage as the transplanted portion is fused comparatively well
with the normal portion since the damaged portion is filled with
transplanted chondrocytes as they are proliferated directly in the
damaged portion. However, there are still pain, after effects, and
economical burden of a patient eventually due to the operation of
twice and the process of operation is also complicated and
difficult since it is necessary to perform operation when
collecting chondrocytes and when transplanting those cultured
externally are transplanted to the portion of articular cartilage
damage.
And there are problems with chondrocytes in that it takes a
considerable amount of time until as much as the cells that are
necessary for transplantation during external culturing of cells
are obtained as the proliferation and growth of the, cells
collected are not active since the chondrocytes collected are
obtained from fully grown adults in most cases; the treatment
itself could not be accomplished if the cells lose the ability to
proliferate at all; and the form of expression of cells is changed
since chondrocytes are cultured externally. And it is of concern
that the life of cartilage made by culturing again fully grown
cells would not be long. And in case of the second operation,
complications such as pain, scar, etc. after operation are
inevitable since it is necessary to perform a serious incision
since no arthroscopic operational methods have been developed
yet.
It has been reported that there has been a method of obtaining
mesenchymal stem cells (MSCs) that are precursor cells of
chondrocytes and osteoblasts from mesenchymal tissues such as
autologous bone marrows, muscles, skin, etc., proliferating them ex
vivo, and injecting them into the portion of articular cartilage
damage together with polymers.
It is shown that the cell proliferation ability in the method of
treatment of cartilage damage by using mesenchymal stem cells
obtained from grown individuals as described in the above is
somewhat higher than that of the autologous chondrocyte
transplantation method since more undifferentiated cells are
obtained and cultured ex vivo. However, the above-described method
still shows a weak ability to proliferate cells for the treatment
of various ways of cartilage damage fully. Also, the
above-described method is required to have a difficult process of
collection of bone marrows, and is limited in that construction of
an infrastructure such as bone marrow storage banks, etc. is
weak.
As described in the above, the methods of treatment of articular
cartilage damage developed up to the present time have been
problematic in view of their operational processes and effects.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a
composition for the treatment of articular cartilage damage or loss
or defect composed of cellular components separated, proliferated,
and/or differentiated from the umbilical cord blood, and
biocompatible polymers that can have superior effects of treatment
of articular cartilage damage through relatively simple
operations.
The composition for the treatment of articular cartilage damage of
the present invention is characterized by that it is composed of
cellular components separated, proliferated, and/or differentiated
from the umbilical cord blood, and their media. Another composition
for the treatment of articular cartilage damage or loss or defect
of the present invention is characterized by that it is composed of
the above cellular components, their media, and biocompatible
polymers. Still another composition for the treatment of articular
cartilage damage or loss or defect of the present invention is
characterized by that it is composed of cellular components
separated or differentiated from the umbilical cord blood, and
biocompatible polymers.
The cellular components of the composition for the treatment of
articular cartilage damage or loss or defect of the present
invention are one or more cellular components such as mesenchymal
stem cells and/or mesenchymal stern/progenitor cells separated
and/or cultured from the umbilical cord blood, precursor cells that
are differentiated from the above mesenchymal stem cells and/or
stem/progenitor cells, chondrocytes and/or osteoblasts that are
differentiated from the above umbilical cord blood-derived
mesenchymal stern/progenitor cells.
In the composition for the treatment of articular cartilage damage
of the present invention, the umbilical cord blood which is the
originating tissue of cellular components is defined to be the
blood collected from the umbilical vein connecting placenta and
fetus.
Among the cellular components of the composition for the treatment
of articular cartilage damage of the present invention, mesenchymal
stem cells separated from the umbilical cord blood may be
differentiated into mesenchymal tissues such as bones, cartilage,
fatty tissues, muscles, tendon, etc. under proper conditions for
differentiation since they are multipotent contrary to typical
stromal cells of bone marrows. Also, mesenchymal stem cells
originated from the umbilical cord blood are the cells that may be
proliferated under proper conditions without being differentiated
into specific cells or tissues since they have the ability to
self-renewal.
Among the cellular components of the composition for the treatment
of articular cartilage damage of the present invention, precursor
cells include all those that may be obtained during the process of
differentiation of mesenchymal stem cells of the umbilical cord
blood into chondrocytes or osteocytes, chondroblasts, osteoblasts,
etc. among the cells originated from mesenchymal stem cells of the
umbilical cord blood.
The cellular components of the composition for the treatment of
articular, cartilage damage of the present invention may have a
much superior ability to proliferate and differentiate since they
are the cells originated from younger cells compared to the cells
originated from cells including mesenchymal stem cells separated
from adult tissues such as bone marrows, muscles, skin, etc.
In the collection and acquisition of originating tissues of the
cellular components of the composition for the treatment of
articular cartilage damage of the present invention, it is much
easier to collect the cellular components than to collect cells
from adult tissues such as bone marrows, etc. that require for
operative procedures.
Moreover, it is easy to find a donor for the umbilical cord blood
since banking or storing of umbilical cord blood is more feasible
at birth and the stored umbilical cord blood can be used easily
after thawing. Routine storing of bone marrow like umbilical cord
blood is impossible.
And since the cellular components of the composition for the
treatment of articular cartilage damage of the present invention
are the cells of which major histocompatibility antigen HLA-DR
(Class II), which is the most important cause of rejection reaction
in interpolation or organ transplantation, is not expressed, the
autologous umbilical cord blood as well as allogenic umbilical cord
blood may be used in that it is possible to avoid bringing about or
minimize immune reactions such as rejection reaction, etc. that may
be the problem of the conventional transplantation operation.
The media of the composition for the treatment of articular
cartilage damage of the present invention is for suspending
cellular components. Generally used cell culture media such as
McCoys 5A media (Gibco), Eagle's basal media, CMRL media, Glasgow
minimum essential media, Ham's F-12 media, Iscove's modified
Dulbecco's media, Liebovitz' L-15 media, RPMI 1640 media, etc. may
be used.
In the present invention, if necessary, one or more secondary
components may be added to the cell culture media. That is, one or
more components selected from the sera of a fetal calf, horse,
human, etc.; antibiotics such as Penicillin G, gentamycin,
streptomycin sulfate, etc. for preventing contamination of
microorganisms; antifungal agents such as amphotericin B, nystatin,
etc. may be used.
Biocompatible polymers of the composition for the treatment of
articular cartilage damage of the present invention are
characterized by having one or more characteristics among the
biocompatibility, biodegradation property and ability to enhance
nutrition of cells and ability to enhance formation of
intercellular substrate. Biocompatible polymers of the composition
for the treatment of articular cartilage damage of the present
invention have the semi-solid or gel-like property to the degree
that is similar to that of ointments or pastes as well as the
mechanical strength and flexibility to the degree that are proper
for cellular transplantation and regeneration of cartilaginous
tissues or bone.
Accordingly, the composition for the treatment of articular
cartilage damage of the present invention is able to accelerate
proliferation and differentiation of chondrocytes that are
transplanted together as it is located at the damaged region,
continuously while maintaining a constant shape once it is
transplanted and seeking for convenience in operation since its
shape may be changed readily to conform to various 3-dimensional
geometry that may be shown in the damaged region of cartilage.
Biocompatible polymers that may be used for the composition for the
treatment of articular cartilage damage of the present invention
may be one or more components selected from natural polymers such
as proteins, polysaccharides, etc; synthetic polymers comprised of
hydroxy acids or their derivatives; and organic polymers forming
the 3-dimensional scaffold or lattice structure according to
chemical binding and their derivatives and transformed compounds.
For example, the proteins among natural polymers include fibrin,
gelatin, and collagen; saccharides among natural polymers include
hyaluronic acid, etc.; synthetic polymers include polyphosphazine,
polyacrylate, polyglactic acid, and polyglycolic acid; and organic
polymers include pluronic acid, alginic acid and its salts,
etc.
It is possible to promote the mechanical strength and flexibility
of the composition for the treatment of articular cartilage damage
of the present invention by adding the chitosan fiber.
If a polymer is used singly in the present invention, it is
possible to adjust its concentration according to the molecular
weight and characteristics of each polymer component. The final
content of pluronic acid after it is mixed with cells when it is
used singly is 30%, and it is preferable to make the final content
of hyaluronic, acid 3-4% after it is mixed with cells when it is
used alone.
When using polymers mixedly in the present invention, it is
preferable to use those that are made by mixing them in such a way
that the final content of pluronic acid after it is mixed with
cells is 15-30% and the final content of hyaluronic acid is
2-4%.
If the chitosan fiber is added to the mixed polymer of the above
pluronic acid and hyaluronic acid in the present invention, it is
preferable to add and mix the same amount of the chitosan fiber to
and with the above mixed polymer.
Illustrated below are the method of manufacture of the composition
for the treatment of articular cartilage damage and the method of
treatment of articular cartilage damage using the above of the
present invention.
The method of manufacture of the composition for the treatment of
articular cartilage damage of the present invention is comprised of
the steps of collection of the umbilical cord blood; separation,
culturing, and/or differentiation of mesenchymal stem cells from
the umbilical cord blood; and mixing those mesenchymal stem cells
and polymers. Each step is further described in detail as
follows:
In the step of collection of the umbilical cord blood, in case of
the normal vaginal delivery, the umbilical cord blood is collected
from the umbilical vein that is extracted fully to the outside in a
state that the placenta remains in the uterus after childbirth, or
from the umbilical vein in a state that the placenta is extracted
fully from the uterus also after childbirth in case of cesarean
section.
When collecting the umbilical cord blood from the umbilical vein
extracted fully to the outside of the uterus after childbirth in
the present invention, it is collected from the umbilical vein
connecting the placenta and fetus after a infant is born according
to aseptis, where both methods of collecting the umbilical cord
blood before the placenta is removed in the uterus after childbirth
and of collecting the umbilical cord blood externally after the
placenta is removed may be used. After the umbilical vein is
secured, the umbilical cord blood is collected in a collection bag
containing an anticoagulant by using collection needles.
All conventional methods including that in Korean Patent
Application No. 10-2002-0008639, and those in Pittinger M F, Mackay
A M, et al. Science 1999; 284: 143-7, Lazarus H M, Haynesworth S E,
et al., Bone Marrow Transplant 1995; 16: 557-64 may be used for the
method of separation and culturing of mesenchymal stem cells and
mesenchymal stem/progenitor cells from the umbilical cord blood
collected as described in the above. Among them, an example is
described as follows:
Mononuclear cells are separated through centrifugal separation of
the umbilical cord blood and washed several times in order to
remove foreign materials. If they are plated and cultured in a
culture dish and/or flask and/or other container after they are
washed, cells are proliferated with forming monolayer. Among them,
mesenchymal stem cells and/or stem/progenitor cells are those of
which shape observed through a inverted microscope is homogeneous
and that are proliferated in the form of colonies of long cells of
the spindle shape. Thereafter, when the cells are grown to be
confluent, sub-culturing is performed in order to have the cells
proliferated until the necessary number of cells is reached.
Mesenchymal stem cells and stem/progenitor cells originated from
the umbilical cord blood of the present invention may be used
directly for the operation or after they go through the
differentiation process.
As to the method of differentiation of mesenchymal stem cells
originated from the umbilical cord blood of the present invention,
any proper method in which desired cells may be obtained may be
selected and used among conventionally used methods (Barry F,
Boynton R E, et al., Exp cell Res 2001; 268: 189-200, Jaiswal N,
Haynesworth S E, et al., J Cell Biochem 1997; 64: 295-312). Among
them one example is described as follows:
While culturing cells originated from the umbilical cord blood in
proper chondrogenic differentiation media or osteogenic
differentiation media, to what degree differentiation is progressed
is confirmed through the measurement of expression of enzymes,
immune expression type analysis, histochemical stain,
histoimmunologic stain, molecular biological examination, or
cellular medium analysis. Mesenchymal stem cells thus manufactured
and the cells differentiated from then may be used directly for the
operation, or may be kept frozen, thawed when necessary, and
proliferated again to be used.
The method of keeping the cells of the present method frozen is
performed according to the widely known method (Doyle et al.,
1995). The medium used for keeping frozen is composed of 10-20% FBS
(fetal bovine serum), 10% DMSO (dimethylsulfoxide), and 5-10%
glycerol. The cells are suspended in such a way that about
1.times.10.sup.6 to 5.times.10.sup.6 cells exist in 1 ml of the
above medium.
The above suspension of cells is distributed into glass or plastic
amples for low-temperature freezing, sealed, and put into a
controlled rate freezer with the conditions for temperature
adjusted in advance. It is preferable to use a freezing program
offering the change of temperature of -1.degree. C./min when
freezing the cells since it is possible to reduce damage to the
cells when they are thawed thereafter. Once the temperature of
amples reaches -180.degree. C., they are transferred to a liquid
nitrogen storage tank. The cells kept frozen may be stored for
several years. Whether the viability of the cells is maintained
should be checked periodically at least every five years. When
thawing the cells kept frozen, the amples are moved promptly to a
water tub of which temperature is adjusted to 37.degree. C. from
the liquid nitrogen storage tank. The content thawed in the amples
is moved immediately to a culturing container having the medium
containing 10% FBS and 5% ES in the sterilized state. The density
of cells on the culturing medium is adjusted to have about
3.times.10.sup.5 to 6.times.10.sup.5 cells existed per ml of the
medium. Whether the cells are proliferated is checked with a
inverted microscope every day. When a proper density of cells is
reached, the cells are transferred to a new medium for
sub-culturing.
The cells cultured as described in the above are transplanted
directly to the region of articular cartilage damage in the state
suspended on a proper medium or after they are mixed with polymers.
In other words, a method of injection of the cells suspended on the
medium to a space made of appropriate biomembranes such as
periosteum, etc. of the region of cartilage damage in a state not
mixed with polymers, sealing them so that the suspension of cells
is not leaked out through the cracks of this periosteum, and
suturing all of incised sections of operation may be used. Or the
above cells may be used by mixing them with appropriate polymers
along with the medium and making them in a state which is similar
to that of ointments or pastes. In all cases, it is preferable to
adjust the concentration of cells contained finally in the
composition of the present invention to have 1.times.10.sup.6 to
5.times.10.sup.7 cells existed in 1 ml. The amount of
administration of the composition of the present invention may be
increased or reduced according to the size of the portion of
articular damage to be cured, and generally, it is preferable to
use about 2 ml in case of knee joints of adults having the size of
about 2 cm.sup.2.
In the method of treatment of the articular cartilage damage using
the composition of the present method, the articular cartilage
portion to be operated is observed preferably through the
arthroscopic operation, the damaged portion is prepared to
facilitate the operation, the composition of the present invention
is applied to the damaged portion, and whether it is positioned
stably is confirmed preferably by using an arthroscope. The form of
the composition of the present invention may be formulated to fit
into the damaged portion in advance before it is applied to the
lesion or changed to fit after it is applied into the damaged
portion.
As described above, the method of treatment of the articular
cartilage damage using the composition of the present invention may
secure the convenience of the procedures and reduce pain, sequelae
and morbidity of a patient since the damaged cartilage may be cured
sufficiently through, the arthroscopic operation, whereas
conventional cell plantation methods require for the operation of
several times.
BRIEF DESCRIPTION OF THE DRAWINGS
The file of this patent contains at least one drawing executed in
color. Copies of this patent with color drawing(s) will be provided
by the Patent and Trademark Office upon request and payment of the
necessary fee
The foregoing and other objects, aspects, and advantages will be
better understood from the following detailed description of a
preferred embodiment of the invention with reference to the
drawings, in which:
FIGS. 1a and 1b are the diagrams in which the viscosity and
strength of a polymer manufactured by mixing pluronic acid and
hyaluronic acid, and that manufactured by mixing the chitosan
fiber, pluronic acid, and hyaluronic acid are confirmed;
FIGS. 2a and 2b are the diagrams showing the results of treatment
of the portion of articular cartilage damage of rabbits by using
the composition of the present invention; and
FIGS. 3a and 3b are the diagrams showing the amplified portion of
cartilage produced newly by the composition of the present
invention.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION
Hereinafter, the present invention is illustrated in more detail in
a preferred embodiment of the invention. However, the scope of the
present invention is not limited by that preferred embodiment and
it could be changeable for human application.
Referring now to the drawings, illustrated in the following
preferred embodiment are the methods of manufacture of the
composition of the present invention and of treatment of the
articular cartilage damage using the above.
Firstly, cellular components are separated and cultured as follows
in order to manufacture the composition of the present
invention:
Mononuclear cells are separated through centrifugal separation of
the umbilical cord blood and washed several times in order to
remove foreign materials. If they are plated and cultured in a
culture dish and/or flask and/or other container after they are
washed, cells are proliferated with forming monolayer. Among them,
mesenchymal stem cells and/or stern/progenitor cells are those of
which shape observed through a inverted microscope is homogeneous
and that are proliferated in the form of colonies of long cells of
the spindle shape. Thereafter, when the cells are grown to be
confluent, sub-culturing is performed in order to have the cells
proliferated until the necessary number of cells is reached.
Mesenchymal stem cells and/or stern/progenitor cells of the
umbilical cord blood obtained as described in the above are treated
with trypsin, washed, suspended on a DMEM medium, and prepared for
so that they may be mixed with the polymer in subsequent steps.
Secondly, for the manufacture of the composition of the present
invention, polymers are prepared as follows and chemical,
biological and/or mechanical properties of polymers are confirmed
by performing experiments confirming their chemical, biological
and/or mechanical strength in advance.
In order to confirm the mechanical strength according to the
concentration or addition or reduction of the components comprising
polymers, a polymer containing 30% of pluronic acid finally, a
polymer containing 4% of hyaluronic acid finally, a polymer
containing hyaluronic acid of each different concentration while
containing 15% of pluronic acid finally, and a polymer containing
the mixture of the above pluronic acid and hyaluronic acid and
further containing the same amount of the chitosan fiber are
prepared, in which the final concentrations of hyaluronic acid are
0.5%, 1.0%, 1.5%, 2.0% and 4.0%, respectively.
Each polymer is prepared by mixing the above components and whether
each has the proper/appropriate property is evaluated.
As a result, as shown in FIG. 1a, a fairly good mechanical strength
is shown in case of the polymer containing hyaluronic acid of each
different concentration while containing 15% of pluronic acid
finally. But it is seen that the mechanical strength is lowered as
the concentration of hyaluronic acid is lowered. Particularly, it
is observed that the polymer flows down along the wall of the tube
if the tube is held upside down when less than 0.5% of hyaluronic
acid is contained finally as the mechanical strength is lowered
significantly.
On the other hand, the polymer containing the mixture of the above
pluronic acid and hyaluronic acid and additionally the same amount
of the chitosan fiber shows the flexibility of the degree which is
good to change the shape when it is applied to the damaged region
while maintaining the mechanical shape irrespective to the amount
of hyaluronic acid. The polymer containing 30% of pluronic acid
finally also shows proper mechanical strength and flexibility.
Accordingly, it is possible to select a polymer having the
appropriate strength and flexibility to the degree which is proper
for applying to the treatment of the region of cartilage damage and
its mixed composition.
Thirdly, in order to manufacture the composition of the present
invention, among the polymers manufactured in the above, for
example, a polymer containing 30% of pluronic acid finally is
selected, of which 0.3 g is mixed with about 0.9 ml of the DMEM
medium sufficiently. Thereafter, the suspension of cells in the
first step in which 1.times.10.sup.7 cells are contained is
concentrated in terms of centrifugal separation, the supernatant is
discarded, and the polymer made in the second step is put into the
cell portion only. A proper amount of the medium is then added to
the above to make the final volume of 1 ml. That is, the
composition for the treatment of articular cartilage damage of the
present invention is manufactured to include 30% (0.3 g) of the
polymer and 1.times.10.sup.7 cells in 1 ml of the medium. If the
amount becomes greater, they are mixed at the same ratio.
Fourthly, the articular cartilage damage is treated by using the
composition for the treatment of articular cartilage damage of the
present invention.
In order to design models for the articular cartilage damage, a
healthy rabbit is selected and proper amounts of ketamine (35
mg/kg) and xylazine (5 mg/kg) according to its body weight are
injected intramuscularly. After it is confirmed that the anesthesia
of the rabbit is completed fully, the knee joints of both legs are
shaved and fixed with an adhesive plaster while maintaining the
supine posture. Both knee joints are disinfected with betadine,
their locations are confirmed by palpation with fingers, after
which the inside of the joint is observed by reaching the inside of
knee joints through the paramedian approach along the incision line
passing through the upper and lower portions of knee joints and
inner side of the knee cap and bending the knee joints while
pushing the knee cap toward the outer side.
After it is confirmed that there are no particular pathologic
findings, a centralizing dimple is made at a position 4 mm above
the front upper end of the central interchondylar notch of the
distal femur with a sharp-pointed gimlet and a hole having the
diameter of 3 mm and depth of 3 mm is made centering around the
scar with a drill. A punch having the diameter of 3 mm may be used
for the same region, and the fall thickness cartilage defect is
created. The damaged region is observed 3-4 months after the
cartilage damage is induced as in the above. And it is confirmed
that the damaged region of cartilage is not cured by itself.
In the meantime, 0.25 ml of the composition of the present
invention manufactured in the third step is injected and pushed
into the damaged region of cartilage of a rabbit by using a
syringe.
Thereafter, the knee cap is returned to its original position, soft
tissues around the knee cap are repaired with absorbable sutures,
and the skin is closed with non-absorbable sutures. The leg on the
opposite side is regarded to be a control group, into which only
biodegradable polymers are applied.
After it is confirmed that the rabbit regains consciousness from
anesthesia, it is allowed to move freely, and antibiotics are
administered to it in order to prevent infection after the
operation until the next day. After 11 weeks, sections of the
articular cartilage region of each rabbit which were subject to
damage and treated are obtained and inspected for newly formed
cartilage. The results of comparison are shown in FIGS. 2a and
2b.
As shown in FIGS. 2a and 2b, the total thickness of repaired layers
produced newly after the composition of the present invention is
applied is shown to be more than twice greater (FIG. 2b) than that
when only the polymer is applied (FIG. 2b).
FIGS. 2a and 2b are amplified and shown in FIGS. 3a and 3b in order
to compare the property of newly formed cartilage repair tissue. As
shown in FIGS. 3a and 3b, cartilage tissue of the almost same
appearance as that of original normal outside cartilage tissue
(right side) are produced newly (left side) if the composition of
the present invention is applied (FIG. 3b), although the cellular
density is greater than that of original normal cartilage tissues.
On the other hand, if only the polymer is applied (FIG. 3a), the
shape of cells is rough compared to that of normal cells and the
density is shown to be low.
Accordingly, it is seen that the composition of the present
invention is able to produce cartilage tissue in the damaged region
of articular cartilage with a superior efficiency and to treat the
articular cartilage damage effectively.
In conclusion, the composition for the treatment of articular
cartilage damage of the present invention shows excellent effects
in histologic aspects for the treatment of the cartilage damage.
Compared to the conventional methods of treatment of the articular
cartilage damage, the method of its treatment using the composition
of the present invention is able to reduce the time, efforts, and
expenses for the treatment of articular cartilage damage since the
subject method has an effect of sufficiently repairing the
articular cartilage damage by employing simple procedures such as
the arthroscopic operation or much simpler operation.
While the invention has been described in terms of a single
preferred embodiment, those skilled in the art will recognize that
the invention can be practiced with modification within the spirit
and scope of the appended claims.
* * * * *